The RAS genes (KRAS, HRAS, and NRAS) are well-known oncogenes, and mutations in these genes can lead to uncontrolled cell proliferation and metastasis, causing cancer [1]. The three human RAS genes encode four RAS proteins: HRAS, NRAS, KRAS4A, and KRAS4B [2]. RAS proteins, a class of intrinsic GTP-binding proteins, act as binary molecules that switch signaling pathways by dynamically cycling between the inactive GDP-bound and active GTP-bound states, thereby regulating cell proliferation differentiation [3]. The RAS proteins exhibit weak endogenous GTPase activity, converting GTP into GDP. GTPase-activating proteins (GAPs) regulate this biochemical process, facilitating the transition of RAS from its active GTP-bound state to the inactive GDP-bound state by enhancing its intrinsic GTPase activity [4]. After activation, RAS will trigger downstream signaling proteins, including RAF protein kinase and phosphoinositide 3-kinase (PI3K) [5,6].

The MAPK (RAS-RAF-MEK-ERK) pathway and the PI3K-AKT (RAS-PI3K-AKT-mTOR) pathway are the two pathways closely associated with cell growth, proliferation, differentiation, and survival (Fig. 1E) [7,8]. In the MAPK pathway, RAF proteins, including ARAF, BRAF, and CRAF, activate downstream MEK, which increases ERK1/2 phosphorylation. The activated ERK1/2 then translocates to the nucleus, mediating the transcription of cytokines such as ELK-1, ATF, c-JUN, and c-FOS, and regulating biological processes like cell proliferation, differentiation, apoptosis, and carcinogenesis. Among RAF proteins, BRAF ablation has a minimal effect on tumor progression. In contrast, CRAF ablation prevents tumor development, underscoring its essential role in KRAS signaling and its potential as a therapeutic target [[9], [10], [11]]. In the PI3K-AKT pathway, RAS can interact directly with the catalytic subunit of PI3K, prompting its transfer to the plasma membrane and leading to conformational changes [12,13]. Once activated, PI3K can induce AKT phosphorylation at Thr308 and Ser473. Activated AKT stimulates its substrate, the mammalian target of rapamycin (mTOR), which plays a crucial role in biological processes such as cell proliferation and apoptosis [14].

Under normal physiological conditions, RAS activity is tightly regulated to maintain proper cellular function. However, mutations in the RAS gene, particularly KRASG12C, KRASG12D, and KRASG12V, lead to constant RAS activation, resulting in persistent downstream signaling, uncontrolled cancer cell growth, and evasion of apoptosis. Consequently, targeting mutant KRAS has emerged as a crucial strategy in cancer treatment therapy. While small-molecule inhibitors like Sotorasib [15] and Adagrasib [16] have achieved clinical success, the emergence of resistance has prompted the search for alternative therapeutic approaches. In recent years, peptide inhibitors, including monocyclic peptides, bicyclic peptides, stapled peptides, and proteomimetic peptides, have gained increasing attention as a novel class of therapeutics due to their ability to bind to RAS proteins and disrupt their interactions with downstream effectors, showing promising anticancer potential [17].

Monocyclic peptides are among the most significant inhibitors. For example, in 2020, Suga et al. [18] identified three cyclic peptides using the Random non-standard Peptides Integrated Discovery (RaPID) platform and assessed their impact on binding to KRASG12D using time-resolved fluorescence energy transfer (TR-FRET) assay. These peptides effectively inhibit the interaction between KRASG12D-GppNHp and RAF1-RBD at micromolar concentrations (IC50 value: KD1 = 3.0 μM, KD2 = 12.5 μM, KD17 = 15.3 μM). However, their poor cell permeability significantly limits their activity within cell assays. Similarly, through phage display screening, Tani et al. [19] identified KRpep-2 and confirmed its high binding affinity to KRASG12D (Kd = 51 nM) via SPR analysis. After optimization of KRpep-2, they obtained KRpep-2d with improved binding affinity and cellular activity. However, they concluded that the inadequate in vivo efficacy resulted from the weak cell membrane permeability. Consequently, they attached at the N-terminal of KRpep-2d to enhance the permeability and resulted in non-specific binding and strong cytotoxicity. Bicyclopeptide, Cyclorasin B3, displayed high binding affinity in the fluorescence anisotropy assay (Kd = 1.2 μM, GTP-bound KRAS) [20]. However, its poor membrane permeability resulted in modest cellular activity. Its optimized derivative, Cyclorasin B4-27 [21], significantly enhanced RAS binding affinity while improving cellular permeability, mainly exhibiting strong antiproliferative activity in KRASG12V-mutant cells (Kd = 21 nM, GTP-bound KRAS; (IC50 ≈ 3 μM, H358 cell line). Additionally, proteomimetic peptides, designed to mimic the structure of protein domains, compete with natural proteins for binding to RAS and have emerged as a promising new class of RAS inhibitors. For example, CHDSOS-5 (Kd = 2.0 ± 0.3 μM), designed by the Arora group in 2021 [22], mimics the helix-loop-helix structure of the RAS-SOS complex and has demonstrated high binding affinity and conformational stability. However, the complexity of its synthesis remains a significant drawback, and the peptide's poor metabolic stability and its limited cellular penetration hamper its practical use. While proteomimetic peptides present an exciting strategy for targeting RAS proteins, their broad clinical application remains a distant goal due to these challenges.

Additionally, stapled peptides have received considerable attention for their capacity to improve stability and biological activity through structural modifications. For instance, HBS3, developed by Bar-sagi and Arora [23], effectively inhibits the RAS-SOS interaction. The binding affinity (Kd, evaluated by fluorescence polarization assay) for the nucleotide-free RAS form is 28 ± 4.8 μM, while for the GDP-bound RAS form, it is 158 ± 16 μM. Recently, α3βHBSSOS, designed by Arora's team [24], showed a significantly enhanced binding affinity with a Kd value of 4.1 ± 2.7 μM, specifically targeting KRASG12C. Additionally, SAH-SOS1<SUB>A</SUB>, introduced by Walensky's group [25], demonstrates potent inhibitory effects on wild-type and mutant KRAS-expressing cancer cells, with an IC50 range between 5 μM and 15 μM. These peptides employ an all-hydrocarbon stapling strategy to stabilize their helical structure, thereby improving their binding affinity for RAS proteins and increasing membrane permeability. However, these peptides still face challenges, including proteolytic instability and insufficient specificity. For example, Bar-sagi and Arora's study indicated that while HBS 3 exhibited good cell membrane penetration, its stability in vivo remained a concern [26].

In summary, while these peptide inhibitors demonstrate considerable potential in RAS-targeted therapy, they all share certain limitations, such as poor membrane permeability, inadequate stability, and issues with specificity. To address these challenges, further chemical modifications and structural optimizations are crucial for improving their therapeutic efficacy and broading their potential in clinical settings.

In 2021, Simanshu et al. [27] investigated the interaction between KRAS and RAF1 and identified the roles of the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF1 in its activation. The cocrystalline structure of wild-type and oncogenic KRAS mutants with CRAF reveal that RBD and CRD act as a unified structural entity. A short linker connects RBD and CRD, and both interact extensively with KRAS. RBD mainly binds to the switch I region of activated KRAS through hydrogen bonds and electrostatic interactions. Meanwhile, CRD performs dual functions during RAF activation. It anchors RAF to the cell membrane by interacting with phospholipids and RAS. Further, it enhances the RAS-RAF association through hydrogen bonds and hydrophobic interactions with other regions of RAS, particularly the inter-switch region and the α5 helix. These complementary roles of RBD and CRD in RAF activation provide pivotal insights into RAS-RAF interactions and offer potential therapeutic targets to disrupt aberrant RAS-RAF interactions.

We speculated that peptides mimicking the key domain of RBD or CRD may disrupt the interactions between RAS and RAF. Herein, we designed and synthesized peptides derived from the α1 helix in the RBD and the η1 helix in the CRD to identify the motif with high binding affinity. Next, we tried a stapling strategy, amino acid mutation, and fatty acid incorporation to enhance the binding affinity, stability, and cellular permeability. Then, we evaluated the anticancer activity and investigated the mechanism in vitro. Two stapled peptides with stearic acid-tag, Sraf-2-1 and Sraf-7-1, showed the potent activities of inhibiting cancer cell proliferation, including melanoma (A375, SK-MEL-2, B16–F10) and lung cancer (A549). Western blot (WB) results revealed that Sraf-2-1 and Sraf-7-1 decreased the expression level of p-AKT concentration-dependently. The membrane permeability of the stapled peptides was improved by conjugating stearic acid to the N-terminus of the stapled peptide, which may contribute to the high cellular activity. The results indicated that the stapled peptides bound to RAS proteins, blocked the PI3K pathway, and induced tumor cell apoptosis as RAS-binding peptide inhibitors. Sraf-2-1 and Sraf-7-1 represent lead peptides for treating RAS-driven cancers.